Sensitive LC Method with Fluorescence Detector for the Determination of Rhodamine 123 in Cell Lysate

Rhodamine 123 has been frequently used to evaluate the functional activity of P-glycoprotein, assess the related drug interactions, analyze mitochondrial distribution and function, sort functionally distinct cell subpopulations and measure mitochondrial or cellular membrane potential. We developed a sensitive and rapid liquid chromatographic method with fluorescence detection after simple sample preparation procedure for the determination of Rhodamine 123 in P-glycoprotein efflux studies. The mobile phase consisted of methanol and 15 mM dibasic sodium phosphate buffer (pH 6.0) (80:20,v/v), delivered at a rate of 1.0 mL min⁻¹. 15 μL of the samples were injected into a reversed-phase C₁₈ column with a fixed excitation wavelength at 505 nm and altered emission wavelengths. The whole LC analysis was accomplished within 6 min. The established linearity range from 1 to 100 ng mL⁻¹, with the inter-day and intra-day RSD below 7.57 and 4.89% at concentrations of 4.4, 44 and 88 ng mL⁻¹. All the calibration standards and quality controls were prepared in cell lysate and were stable for three freeze-thaw cycles, for 12 h at 4 °C and for 6 h at room temperature. This rapid LC method has been applied to the quantification of Rhodamine 123 in cell lysate obtained from P-glycoprotein efflux study conducted in rat brain capillary endothelial cells.